Journal of Animal Science and Technology

  • 제49권5호
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  • Pages.549-558
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  • 2007
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  • 2672-0191(pISSN)
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  • 2055-0391(eISSN)
한국축산학회 (Korean Society of Animal Sciences and Technology)
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돼지 SLA class III 영역 내 C4B 및 BAT2의 cSNP 동정 및 이를 이용한 유전자형 분석

cSNP Identification and Genotyping from C4B and BAT2 Assigned to the SLA Class III Region

  • 김재환 (경상대학교 대학원 응용생명과학부(BK21)) ;
  • 임현태 (경상대학교 대학원 응용생명과학부(BK21)) ;
  • 서보영 (경상대학교 대학원 응용생명과학부(BK21)) ;
  • 이상호 (경상대학교 학교기업 GAST) ;
  • 이재봉 (경상대학교 대학원 응용생명과학부(BK21)) ;
  • 유채경 (경상대학교 농생명학부) ;
  • 정은지 (경상대학교 농생명학부) ;
  • 전진태 (경상대학교 대학원 응용생명과학부(BK21))
  • Kim, J.H. (Division of Applied Life Science(BK21) Graduate School of Gyeongsang National University) ;
  • Lim, H.T. (Division of Applied Life Science(BK21) Graduate School of Gyeongsang National University) ;
  • Seo, B.Y. (Division of Applied Life Science(BK21) Graduate School of Gyeongsang National University) ;
  • Lee, S.H. (Gyeongnam animal science and technology, Gyeongsang National University) ;
  • Lee, J.B. (Division of Applied Life Science(BK21) Graduate School of Gyeongsang National University) ;
  • Yoo, C.K. (Division of Agriculture and Life, Gyeongsang National University) ;
  • Jung, E.J. (Division of Agriculture and Life, Gyeongsang National University) ;
  • Jeon, J.T. (Division of Applied Life Science(BK21) Graduate School of Gyeongsang National University)
  • 발행 : 2007.10.31
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초록

C4B 및 BAT2는 SLA class III 영역에 존재하며, 최근 들어 사람의 질병과의 연관성이 보고되고 있다. GenBank database로부터 수집된 사람과 마우스의 C4B 및 BAT2의 CDS를 염기정렬하여 상동성이 높은 부분에서 primer를 제작한 후 RT-PCR 및 RACE-PCR을 수행하여 돼지 C4B 및 BAT2 유전자의 CDS 서열을 결정하였다. 염기서열이 결정된 돼지 C4B와 BAT2의 CDS 길이가 각각 5226 bp와 6501 bp로 나타났다. 이들 각각의 CDS 및 아미노산 서열을 사람 및 마우스와 비교한 결과 CDS는 76~87%, 아미노산 서열은 72~90%의 상동성을 보였으며, C4B가 BAT2에 비해 다소 낮게 나타났다. 두 유전자에서 나타나는 cSNP를 분석하기 위해서 exon 영역을 증폭하기 위한 primer를 제작하였으며, 돼지 6품종을 대상으로 direct sequencing을 실시하였다. 그 결과 C4B로부터 4개, BAT2로부터 3개의 cSNP가 확인되었다. 또한 7개의 cSNP 중 C4B의 C4248T를 제외한 6개의 cSNP에 의해서 아미노산 치환이 발생하였다. 동일한 DNA를 사용하여 7개의 cSNP를 대상으로 Multiplex-ARMS 방법을 사용하여 유전자형 분석을 실시한 결과 direct sequencing 결과와 일치하였다. Multiplex-ARMS 방법의 재현성을 재확인하기 위해 무작위로 2개의 DNA 시료를 선택하여 direct sequencing과 Multiplex-ARMS 분석을 실시하여 유전자형이 일치함을 다시 확인하였다. 따라서 본 연구에서 확인된 7개의 cSNP는 SLA class III 지역의 haplotype 분석을 위한 기초 자료로 사용될 수 있으며, Multiplex- ARMS 기법은 이종장기 개발에 필수적인 SLA 전체 영역 내 유전자들의 유전자형 분석을 위한 효율적인 분석방법이라고 사료된다.

C4B and BAT2, assigned to the SLA class III region, were recently reported on relation with human diseases. The primers for RT-PCR and RACE-PCR for CDS analysis of these genes of pig were designed by aligning the CDSs of humans and mice from GenBank. After we amplified and sequenced with these primers and cDNAs, the full-length CDSs of pig were determined. The CDS lengths of C4B and BAT2 were shown as 5226 bp and 6501 bp. In addition, the identities of nucleotide sequences with human and mouse were 76% to 87%, and the identities of amino acids were 72% to 90%. After we carried out the alignment with determined CDSs in this study and pig genomic sequences from GenBank, the primers for cSNP detection in genome were designed in intron regions that flanked one or more exons. Then, we amplified and directly sequenced with genomic DNAs of six pig breeds. Four cSNPs from C4B and three 3 cSNPs from BAT2 were identified. In addition, amino acid substitution occurred in six cSNP positions except for C4248T of C4B. By the Multiplex-ARMS method, we genotyped seven cSNPs with DNA samples used for direct sequencing. We verified that this result was the same as that analyzed using direct sequencing. To demonstrate recrudescence, we performed both direct sequencing and Multiplex-ARMS on two randomly selected DNA samples. The genotype of each sample showed the same result from both methods. Therefore, seven cSNPs were identified from C4B and BAT2 and could be used as the basic data for haplotype analysis of SLA class III region. Moreover, the Multiplex-ARMS method should be powerful for genotyping of genes assigned to the whole SLA region for the xenograft study.

키워드

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